Bacterial infection remains a leading cause of morbidity and mortality worldwide, not least due to the unprecedented spread of antibiotic-resistant pathogens. Moreover, effective infection control is hampered by the poor performance of standard diagnostics and hence inadequate choices are being made about the use of effective treatments. This alarming situation is posing an enormous challenge for clinical practice, public healthcare and biomedical research.
Current diagnosis and treatment of suspected infections depends largely on the positive identification of the likely microbiological pathogen, a concept that was introduced by Robert Koch more than a century ago but has not changed since then. Positive identification of the causative pathogen of a bacterial infection is not only crucial to guide and refine patient management with respect to the choice of antibiotic treatment, but it also provides us with important clues as to the underlying inflammatory mechanisms and how these can be manipulated to help resolve infection.
Presently, detection and characterisation of infectious organisms requires classical microbiological culture techniques, spectrometry based assays and/or highly complex nucleic acid identification methods. Mohan et al, 2011 describe the combined use of nucleic acid detection and immunoassay detection e.g. “oligonucleotide probes optimized for hybridization at 37° C. to facilitate integration with the immunoassay” to diagnose urinary tract infections [5]. Podsiadly et al, 2005 describe detection of levels of specific Chlamydia pneumoniae IgM, IgG and IgA serum antibodies to diagnose urinary infections [6]. These latter assays are considered unreliable.
Moreover, pathogen identification is significantly hampered by the inherent delay associated with a reliance on conventional culture techniques. It often takes 2 days, or even longer, before the results are available to the treating doctor, to enable specific and targeted therapy. In some diseases, such as tuberculosis, this can take up to two months. Most importantly, in many cases bacteria cannot be cultured by traditional methods, meaning that the cause underlying the patient's clinical symptoms remains unknown.
Typically, infection is only apparent when significant clinical symptoms appear. Effective and specific therapeutic intervention depends on early detection and identification of the causative organism. Conventionally, patient management is determined on the basis of symptoms, initial clinical findings, and other basic laboratory markers of inflammation such as C-reactive protein. This approach is often sub-optimal being too general and lacking in specificity. Increased specificity is generally only attained by microbiological identification and antibiotic sensitivity testing, and this can be associated with delay in the optimal management of the infection. The tendency to use broad spectrum antibiotics to cover a number of possible aetiological agents encourages the development of other serious problems such as C. difficile infection, and also bacterial resistance. Thus, there is a clear clinical need to achieve earlier and better detection and characterisation of the infection in order to 1) improve patient outcomes; 2) improve the use of specific anti-infectives; and 3) reduce antimicrobial resistance and the development of other serious infections. Technologies capable of achieving this should enhance the patient experience, and ensure a more sustainable, cost-effective approach to patient management.
Peritoneal dialysis (PD) is a treatment for patients with severe chronic kidney disease. Therapy involves the introduction of specialised dialysis fluids into the peritoneal cavity, via a Tenckhoff tube, with the aim of removing toxins that accumulate on kidney failure by diffusion and also water by the osmotic gradient created by the fluid. Both processes occur contemporaneously across the semipermeable peritoneal membrane. Infection of the glucose rich dialysis fluid, leading to peritonitis, is a major complication of PD that has significant morbidity and mortality implications.
Peritonitis can have a detrimental effect on both short and long term patient health. In the short term, severe peritonitis episodes can result in the need to immediately remove the Tenckhoff tube and, therefore, treatment failure. In the long term, recurrent or severe peritonitis can result in thickening (fibrosis) of the peritoneal membrane and significant alterations in membrane permeability/function that lead to treatment failure. Early recognition of the onset of peritonitis coupled with rapid investigation, diagnosis, and effective treatment is imperative for successful outcome. Identification of the causative micro-organism using standard techniques can be difficult due to the relatively low bacterial numbers and the length of time required to culture them (often many days). This leads to the phenomenon of ‘culture negative’ peritonitis, i.e. all clinical and laboratory parameters confirm the diagnosis but no organism is ever identified, in 0-50% of cases [3]. Consequently, given the delays in micro-organism confirmation, all patients are started on dual antibiotic treatment to cover both Gram-positive and Gram-negative bacteria. Only when (and if) the organism is identified is the antibiotic regimen refined. If the PD fluid specimen remains culture negative, then the patient is required to undergo prolonged dual, broad spectrum, antibiotic treatment.
As mentioned above, whilst molecular tests that identify microorganisms in biological fluids are available, they often suffer from a lack of specificity, unacceptably high rates of false positivity and the frequent identification of non-pathogenic species that are due to contamination of the sample or asymptomatic carriage.
It follows from the above that point-of-care methods that direct therapy, especially in cases where organism species or virulence is a determinant of outcome, are urgently required.
The present disclosure relates to methods of rapid diagnosis of infection in peritoneal disease. In the present study we performed a detailed immunological and microbiological analysis in PD patients on the first day of presentation with acute peritonitis. Key to this technology is the discovery that the induction of complex cellular and humoral immune responses rapidly follows exposure of the immune system to an infectious agent. Surprisingly, this response is apparent within hours of the exposure and can be measured in the draining effluent of peritoneal dialysis patients with acute clinical symptoms, at the time of presenting with a characteristic but not diagnostic ‘cloudy bag’. As such, these results have far-reaching implications for differential diagnosis of patients with suspected infections and may help guide patient management through faster biomarker-based diagnostics, better predictive risk modelling and improved targeting of a therapy and its ultimate efficacy.